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Toll-like receptor 9 activation: a novel
mechanism linking placenta-derived
mitochondrial DNA and vascular dysfunction in
pre-eclampsia
CLINICAL SCIENCE, LONDON, v. 123, n. 7, pp. 429-435, OCT, 2012
http://www.producao.usp.br/handle/BDPI/34011
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Clinical Science (2012) 123, 429–435 (Printed in Great Britain) doi:10.1042/CS20120130
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Styliani GOULOPOULOU∗ , Takayuki MATSUMOTO†, Gisele F. BOMFIM‡
and R. Clinton WEBB∗
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Department of Physiology, Georgia Health Sciences University, Augusta, GA, U.S.A., †Department of Physiology and
Morphology, Institute of Medicinal Chemistry, Hoshi University, Tokyo, Japan, and ‡Department of Pharmacology,
Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
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Emerging evidence suggests that in addition to being the ‘power houses’ of our cells, mitochondria
facilitate effector responses of the immune system. Cell death and injury result in the release of
mtDNA (mitochondrial DNA) that acts via TLR9 (Toll-like receptor 9), a pattern recognition
receptor of the immune system which detects bacterial and viral DNA but not vertebrate
DNA. The ability of mtDNA to activate TLR9 in a similar fashion to bacterial DNA stems
from evolutionarily conserved similarities between bacteria and mitochondria. mtDNA may
be the trigger of systemic inflammation in pathologies associated with abnormal cell death.
PE (pre-eclampsia) is a hypertensive disorder of pregnancy with devastating maternal and fetal
consequences. The aetiology of PE is unknown and removal of the placenta is the only effective
cure. Placentas from women with PE show exaggerated necrosis of trophoblast cells, and circulating
levels of mtDNA are higher in pregnancies with PE. Accordingly, we propose the hypothesis that
exaggerated necrosis of trophoblast cells results in the release of mtDNA, which stimulates TLR9
to mount an immune response and to produce systemic maternal inflammation and vascular
dysfunction that lead to hypertension and IUGR (intra-uterine growth restriction). The proposed
hypothesis implicates mtDNA in the development of PE via activation of the immune system and
may have important preventative and therapeutic implications, because circulating mtDNA may
be potential markers of early detection of PE, and anti-TLR9 treatments may be promising in the
management of the disease.
INTRODUCTION
PE (pre-eclampsia) is a pregnancy syndrome that is
defined by the onset of hypertension and proteinuria
after 20 weeks of gestation [1]. It affects every maternal
organ and fetal development, is an important cause of
pre-term delivery in developed countries, and a leading
cause of maternal and fetal morbidity and mortality in
developing countries. One of the main characteristics of
the syndrome is an inability of the trophoblasts to invade
Key words: mitochondrial DNA, placenta, pre-eclampsia, pregnancy, Toll-like receptor 9, vascular dysfunction.
Abbreviations: BP, blood pressure; ERK, extracellular-signal-regulated kinase; IL, interleukin; IUGR, intra-uterine growth
restriction; LPS, lipopolysaccharide; MAPK, mitogen-activated kinase; mtDNA, mitochondrial DNA; ODN, oligodeoxynucleotide;
PAMP, pathogen-associated molecular pattern; PE, pre-eclampsia; SBP, systolic BP; TLR, Toll-like receptor.
Correspondence: Dr Styliani Goulopoulou (email [email protected]).
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Clinical Science
Toll-like receptor 9 activation: a novel
mechanism linking placenta-derived
mitochondrial DNA and vascular
dysfunction in pre-eclampsia
430
S. Goulopoulou and others
the decidual arteries, causing defective placentation,
and reduced placental perfusion and nutrient supply
[2]. Other features of the disease include placental
and systemic oxidative stress, and dysfunction of the
maternal vasculature [3–5]. These are also associated
with reduced placental perfusion. As PE progresses to
a clinical stage, the mother presents with symptoms
such as hypertension, proteinuria, coagulopathy and/or
hepatic dysfunction [6]. In most cases, removal of the
placenta alleviates the clinical symptoms of the disease,
indicating that placenta-derived factors are probably
responsible for the pathogenesis and/or manifestation
of PE.
Components of the immune system have been
detected at the maternal–fetal interface [7,8] and their
function in pregnancy has recently become an emerging
field of investigation in an effort to understand
the role of the immune system in defending the
fetus and the mother from infections. Bacterial and
viral infections are often responsible for pregnancy
complications such as pre-term labour and PE [9,10].
Consequently, several investigations have addressed
the question of how exogenous (viral and bacterial)
products induce poor pregnancy outcomes. In this
Hypothesis article, we address the question of how
endogenous molecules released by the placenta induce
clinical symptoms of PE, such as maternal vascular
dysfunction and hypertension, as well as insufficient fetal
growth.
TLRs (Toll-like receptors) are cellular components
of the immune system that detect conserved sequences
known as PAMPs (pathogen-associated molecular
patterns) [11]. Our main knowledge regarding the role
of TLR signalling in pregnancy derives from studies
in placental explants and trophoblast cells. The human
placenta expresses transcripts for TLR1–TLR10 [7–
12], and placentas from patients with PE show greater
expression of TLR2, TLR3, TLR4 and TLR9 compared
with controls [7,13], indicating that TLR signalling may
be involved in the development of placental deficiencies
and the pathogenesis of PE.
PE is characterized by exaggerated trophoblast
apoptosis and necrosis [14,15], and increased expression
of TLR9 in placental [13] and dendritic cells [16].
Furthermore, pregnancies complicated with IUGR
(intra-uterine growth restriction), a common feature
of PE, show elevated levels of circulating mtDNA
(mitochondrial DNA) [17]. Interestingly, the highest
mtDNA levels were found in the more severe IUGR
subsets that were complicated with maternal PE [17].
On the basis of recent evidence that mtDNA induces
an immune response via activation of TLR9 signalling
pathway [18], we propose the hypothesis that abnormal
trophoblast cell death (i.e. exaggerated necrosis) results in
the release of mitochondrial products, including mtDNA,
which stimulate TLR9 to mount an immune response
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and produce systemic maternal inflammation, vascular
dysfunction and IUGR.
TLR SIGNALLING
TLRs are type I integral membrane glycoproteins that
contain leucine-rich repeats in their extracellular domain
and a cytoplasic TIR [Toll/IL (interleukin)-1 receptor]
signalling domain [19]. These receptors recognize
PAMPs associated with bacteria and viruses, and induce
signals which are critical for eliciting innate and
adaptive immune responses to invading micro-organisms
[11]. In addition to detecting molecular structures
of microbial origin, TLRs respond to endogenous
molecular structures known as DAMPs (damageassociated molecular patterns), which are released due
to cell death and injury [18]. At least 11 TLRs have been
reported in mammals (TLR1–11). TLRs that recognize
constituents of bacterial and fungal cell wall are localized
on the cell surface (TLR1, TLR2, TLR4, TLR5 and
TLR6), whereas those that recognize pathogen-specific
nucleic acids (TLR3, TLR7, TLR8 and TLR9) are
localized to intracellular membranes and bind their
ligands in phagosomes or endosomes [20–23].
TLR9 recognizes bacterial DNA containing the
dinucleotide CG where the C is unmethylated (CpGcontaining DNA) [24]. TLR9 resides in the endoplasmic
reticulum and, upon cell activation with CpG DNA, the
distribution of TLR9 changes, with a portion of the total
protein translocating first into early endosomes and later
into lysosomal compartments, where signal transduction
is initiated [21]. Following CpG DNA binding, TLR9
associates with the intracellular adapter protein MyD88
(myeloid differentiation factor 88) [25] to activate signal
transducing proteins, such as members of the IRAK (IL1-receptor-associated kinase) family, MAPKs (mitogenactivated kinases) or IRFs (interferon regulatory factors)
[26]. These events initiate the synthesis and release of
inflammatory cytokines and antimicrobial products, and
regulate co-stimulatory molecules [25].
The ability of TLR9 to discriminate between foreign
and self-DNA is due to the higher frequency and
presence of unmethylated CpG dinocluoteides in
bacterial and viral compared with mammalian DNA
[27]. Mitochondria, however, evolved from saprophytic
bacteria to become intracellular organelles [28] and,
therefore, mtDNA is structurally similar to bacterial
DNA and shares unmethylated CpG DNA repeats
[29]. Consequently, mtDNA is a ligand for TLR9 [18].
In this Hypothesis article, we propose that mtDNA
released by necrotic trophoblasts induces a maternal
immune response via TLR9 signalling activation, leading
to the development of PE and its associated clinical
symptoms.
TLR9 and vascular function in pregnancy
TLR ACTIVATION AND CLINICAL SYMPTOMS
OF PE
Intrauterine infections are associated with PE in humans
[10,30], and viral and bacterial ligands have been often
used to induce PE-like symptoms in animals. For
instance, pregnant rats infused with low concentrations
of endotoxin (a TLR4 ligand) [31] or treated with
the viral mimetic poly(I:C) (a TLR3 ligand) [32]
developed maternal hypertension, vascular dysfunction
and proteinuria. Endotoxin or poly(I:C) treatment had
no effect in non-pregnant rats [32,33]. Thus activation
of TLR3 and TLR4 causes PE-like symptoms in rats,
providing compelling evidence that viral or bacterial
infection may contribute to the development of the
disease through TLR signalling.
During a systemic or intra-uterine infection in
pregnancy, invading micro-organisms and their breakdown products provide an increased pathogenic load
to the maternal–fetal environment. Hypomethylated
CpG motifs presented by infectious agents may
therefore overstimulate TLR9, mediating maternal
immune activation [34]. Previous studies have examined
the role of the CpG/TLR9 axis in animal models of
pregnancy, focusing on pregnancy outcomes such as
pup survival and development [34,35]. High doses of
a synthetic CpG ODN (oligodeoxynucleotide) during
mouse pregnancy stimulated Th1-cytokine release and
induced fetal resorptions, craniofacial and limb defects,
placental cell necrosis, calcification and inflammation,
suggesting that activation of TLR9 signalling may
have adverse pregnancy outcomes [35]. Thaxton et al.
[34] confirmed these findings and also showed that
anti-inflammatory cytokine proficiency protects against
CpG-induced pregnancy complications. These previous
studies focused on pup survival and growth, but did not
examine maternal physiological functions, which may
be compromised in the presence of an inflammatory
environment.
Preliminary observations in our laboratory suggest
that activation of TLR9 via a synthetic CpG oligonucleotide elicits PE-like symptoms in pregnant rats.
Figure 1(A) shows that continuous activation of TLR9
by exogenous synthetic oligonucleotides increases SBP
[systolic BP (blood pressure)] by ∼20 mmHg in pregnant,
but not in non-pregnant, rats. Furthermore, treatment
with the TLR9 agonist did not affect the number of
pups/litter (13.3 +
− 1.3 compared with 12.7 +
− 0.3 pups
in treated compared with untreated rats respectively),
but reduced fetal weights (Figure 1B). These findings
suggest that activation of TLR9 during pregnancy not
only affects fetal development as reported previously
[34,35], but it also induces maternal hypertension,
which is a main feature of pregnancies with PE. In
contrast with pregnant rats, non-pregnant rats did
not have a hypertensive response to TLR9 activation.
Figure 1 Effect of a synthetic TLR9 ligand on blood pressure
and fetal weight in pregnant rats
A synthetic TLR9 ligand (ODN 2395; InvivoGen) was administered by intraperitoneal
injection (0.1 μg/rat) on days 14, 17, and 19 of gestation (term = 21 days)
in pregnant rats or on the corresponding days in non-pregnant rats. BP was
measured via the tail cuff method on gestational day 20 and rats were killed
on day 21. (A) SBP of non-pregnant (NP) and late pregnant rats (Preg) treated
with ODN 2395 (NP-ODN 2395, n = 1; Preg-ODN 2395, n = 2) or vehicle
(NP-Veh, n = 3; Preg-Veh, n = 2). (B) Fetal weights from pregnant rats treated
with ODN 2395 or vehicle. All procedures were performed in accordance with
the Guiding Principles in the Care and Use of Animals, approved by the Medical
College of Georgia Committee on the Use of Animals in Research and Education
and in accordance with the Guide for the Care and Use of Laboratory Animals
published by the U.S. National Institutes of Health.
Previous studies have shown that the intracellular
localization of TLR9 determines the access of the
receptor to different sources of DNA [20]. It is
unknown, however, whether pregnancy affects TLR9
localization. An increase in TLR9 expression with
gestation could also explain the differential responses to
TLR9 in non-pregnant and pregnant rats. Accordingly,
normal and complicated pregnancies may determine
TLR9 responses to endogenous and/or exogenous threats
by modifying TLR9 localization and expression. The
effects of pregnancy on TLR9 localization and protein
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expression in different cell types are currently under
investigation.
Both bacterial and mtDNA are ligands for TLR9
[18,24]. mtDNA is released from necrotic cells [18]
and circulating levels of mtDNA are elevated in
pregnancies complicated with maternal PE and IUGR
[17]. Furthermore, pregnancies with PE are characterized
by exaggerated trophoblast cell apoptosis and necrosis
[14,15], which may be the source of mtDNA released
in the maternal circulation. Interestingly, smoking is
associated with reduced risk of PE [36] and, although
the exact mechanism of this protection is unknown, it
may relate to the fact that maternal smoking depletes
mtDNA in the placenta [37]. To examine the effects of
TLR9 activation by mitochondrial products on maternal
cardiovascular responses, we injected mitochondria
isolated from rat liver in pregnant rats. Dams injected
with ‘damaged’ mitochondria developed high BP (change
in SBP = 26 mmHg) compared with controls (Figure 2A).
Collectively, previous studies and our preliminary
observations suggest that activation of TLR3, TLR4 and
TLR9 lead to the development of PE-like symptoms
in pregnant animals. Given that TLR9 can be activated
by both bacterial and mtDNA, we suggest that both
endogenous and exogenous DNA leads to PE via TLR9
signalling, and we propose that the source of the
endogenous DNA is mitochondria released by necrotic
trophoblasts.
TLR9 ACTIVATION AND MATERNAL
VASCULAR FUNCTION
Figure 2 Effect of mitochondria on blood pressure, TLR9
protein expression and ERK1/2 phosphorylation when
injected into pregnant rats
Mitochondria were isolated from rat liver (Mitochondria Isolation Kit; Pierce
Biotechnology) and their integrity was disrupted by sonication. Mitochondria
solution (4 mg of tissue/rat, diluted in saline) was injected into pregnant
rats on gestational day 15. BP was measured via the tail cuff method on
gestational day 18 and rats were killed on day 19. (A) SBP of pregnant rats
injected with mitochondria (Preg-mt, n = 2) or Vehicle (Preg-Veh, n = 2).
(B) Densitometric intensity and representative Western blots for TLR9 protein,
in relation to β-actin, in second-order mesenteric arteries from pregnant rats
injected with mitochondria (Preg-mt, n = 2) or Vehicle (Preg-Veh, n = 2). (C)
Densitometric intensity and representative Western blots for phospho-ERK1/2, in
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Maternal vascular dysfunction is a hallmark of PE
that may significantly contribute to the manifestations
of the disease, such as maternal hypertension and
IUGR [38]. Furthermore, PE poses a risk of future
maternal cardiovascular disease [39], which may stem
from alterations in the function of the vasculature during
pregnancy. The exact mechanisms, by which placentaderived factors cause maternal vascular dysfunction, are
currently unknown.
Sustained maternal immune system activation via a
TLR3 ligand [poly(I:C)] during rat pregnancy resulted in
reduced endothelium-dependent conduit artery dilation
[32], indicating that TLR signalling plays a role in the
development of maternal vascular dysfunction in
relation to total ERK1/2, in mesenteric arteries from pregnant rats injected with
mitochondria (Preg-mt, n = 2) or vehicle (Preg-Veh, n = 2). All procedures
were performed in accordance with the Guiding Principles in the Care and Use
of Animals, approved by the Medical College of Georgia Committee on the Use of
Animals in Research and Education and in accordance with the Guide for the
Care and Use of Laboratory Animals published by the U.S. National Institutes of
Health.
TLR9 and vascular function in pregnancy
pregnancies with PE. A recent study showed that infusion
of LPS (lipopolysaccharide; a TLR4 ligand) on gestational
day 15 decreased myogenic tone and increased wall
thickness of posterior cerebral arteries in pregnant,
but not in non-pregnant, rats [33]. These investigators
did not examine the role of TLR4 signalling in LPSmediated vascular effects, but previous studies have
provided compelling evidence that LPS-induced signal
transduction is mediated by TLR4 [40]. In addition,
injection with poly(I:C) (a TLR3 ligand) in pregnant rats
[32] and mice [41], and infusion of LPS in pregnant
rats [33] resulted in increased serum concentrations and
vascular mRNA levels of pro-inflammatory cytokines
respectively. Furthermore, it has been reported that
exogenous IL (interleukin)-10 treatment in pregnant
mice injected with poly(I:C) prevented maternal
endothelial dysfunction [41]. These findings show that
the effects of TLR signalling on maternal vascular
function are probably mediated by the induction of
pro-inflammatory cytokines and can be regulated by antiinflammatory cytokines, such as IL-10.
TLRs are expressed on immune [42] and trophoblast
cells [13], but have been also detected in the vascular
endothelial [43] and smooth muscle [44] cells. Our
laboratory has shown recently that treatment with
an anti-TLR4 antibody reduced BP and small vessel
contractility via a COX (cyclo-oxygenase)-dependent
mechanism in a rat model of hypertension [45],
implicating TLR signalling in hypertension-associated
vascular dysfunction. Studies on TLR signalling in
pregnancy suggest that the vascular effects of TLR
activation are due to an increase in pro-inflammatory
cytokines [32,33,41]. There is a possibility, however, that
exogenous and endogenous ligands directly act on TLRs
in the vascular wall, inducing a cytokine-independent
signalling pathway that leads to vascular dysfunction.
In immune cells, activation of TLR9 via mtDNA and
bacterial DNA induces an immune and inflammatory
response via activation of p38 MAPK [18]. Other
studies suggest the involvement of other MAPKs, such
as ERK (extracellular-signal-regulated kinase) and JNK
(c-Jun N-terminal kinase), in the downstream TLR9
signalling pathway [46,47]. To examine the effects of
TLR9 activation on the activity of ERK1/2 in maternal
resistance vessels, we measured protein expression
of TLR9 and phospho-ERK1/2 in mesenteric arteries
from pregnant rats treated with mitochondria isolated
from rat liver and from rats treated with vehicle.
Expression of TLR9 and phospho-ERK1/2 were greater
in mitochondria-treated pregnant rats compared with
controls (Figures 2B and 2C). Given that ERK1/2 plays a
significant role in vascular responses to constrictor stimuli
(i.e. phenylephrine and thromboxane mimetics) [48,49],
we speculate that ERK1/2 is a downstream effector of the
CpG/TLR9 axis in maternal vascular tissue, mediating a
direct effect of TLR9 ligation by mtDNA on maternal
Figure 3 Overview of the hypothesis that placenta-derived
mtDNA induces PE-like symptoms
During pregnancy, placenta-derived mDNA, through TLR9 activation, increases
vascular reactivity to constrictor stimuli via an ERK1/2-dependent signalling
pathway and potentiates the release of pro-inflammatory cytokines, contributing
to PE-like symptoms (i.e. maternal hypertension and IUGR).
vascular function. Indeed, there are reports to suggest
that activation of TLR9 leads to ERK1/2 activation in
various cells [50].
According to our preliminary results, we propose that
during pregnancy activation of the CpG/TLR9 axis via
mtDNA released by necrotic placental cells increases the
activation of ERK1/2, contributing to increased maternal
vascular reactivity to constrictor stimuli, increased
peripheral vascular resistance, maternal hypertension
and insufficient uterine blood flow (Figure 3). This
mechanism may be independent of the effects of proinflammatory cytokines released upon TLR9 activation
on the maternal vasculature, but this speculation warrants
further investigation. Integrative approaches including
physiological, pharmacological, biochemical, molecular
and cellular techniques, as well as translational studies,
are required to test the proposed hypothesis and
to investigate the role of the innate immune system
in maternal vascular inflammation and dysfunction,
and its contribution to the development of maternal
hypertension and IUGR. Studies of maternal vascular
reactivity, BP responses, uterine blood flow, levels of
maternal proteinuria and fetal development in pregnant
animals treated with mtDNA isolated from placentas
and use of Tlr9-knockout mice can establish a causal
relationship between mtDNA and PE-like symptoms.
Furthermore, cell culture studies can provide information
regarding the direct effects of mtDNA on TLR9
signalling in vascular smooth muscle and endothelial cells.
In addition, assessment of circulating mtDNA content
in blood from women with PE is necessary to verify the
relevance of the proposed hypothesis to pregnancies with
PE.
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PERSPECTIVES AND CLINICAL
IMPLICATIONS
mtDNA is released by necrotic cells inducing a
systemic inflammatory response via activation of TLR9
[18]. Furthermore, under certain pathophysiological
conditions, the ability of TLR9 to discriminate
between self and foreign DNA can be circumvented
and this leads to immune pathologies and chronic
inflammation [51]. In the present article, we propose
the hypothesis that mtDNA is a placenta-derived factor
with immunostimulatory properties that is secreted in the
maternal circulation as a result of exaggerated trophoblast
necrosis, activating the maternal immune system via
TLR9 signalling. These events lead to systemic maternal
inflammation and vascular dysfunction, hypertension
and IUGR. The proposed hypothesis implicates mtDNA
in the development of PE via activation of the
immune system and may have important preventative
and therapeutic implications. For instance, circulating
mtDNA may be potential markers of early detection of
PE, and anti-TLR9 treatments may be promising in the
management of the disease.
FUNDING
This study was supported in part by the National
Institutes of Health [grant numbers R01 HL071138,
R01 DK083685, T32 HL066993-09], the Society for
Women’s Health Research, and the Naito Foundation
Japan.
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Received 7 March 2012/2 April 2012; accepted 5 April 2012
Published on the Internet 7 June 2012, doi:10.1042/CS20120130
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